Increase In Transepithelial Resistance Research Articles

PAI-1 regulates AT2-mediated re-alveolarization and ion permeability

BackgroundAcute lung injury is characterized by overwhelmingly elevated PAI-1 in both lung edema fluid and the circulating system. The role of increased PAI-1, encoded by Serpine1 gene, in the regeneration of injured lung epithelium has not been understood completely. This study aimed to investigate the role of Serpine1 in the regulation of alveolar type 2 epithelial cell (AT2) fate in a humanized mouse line carrying diseased mutants (Serpine1Tg).MethodsWild-type (wt) and Serpine1Tg AT2 cells were either cultured as monolayers or 3D alveolospheres. Colony-forming assay and total surface area of organoids were analyzed. AT1 and AT2 cells in organoids were counted by immunohistochemistry and fluorescence-activated cell sorting (FACS). To test the potential effects of elevated PAI-1 on the permeability in the epithelial monolayers, we digitized the biophysical properties of polarized AT2 monolayers grown at the air–liquid interface.ResultsA significant reduction in total AT2 cells harvested in Serpine1Tg mice was observed compared with wt controls. AT2 cells harvested from Serpine1Tg mice reduced significantly over the wt controls. Spheroids formed by Serpine1Tg AT2 cells were lesser than wt control. Similarly, the corresponding surface area, a readout of re-alveolarization of injured epithelium, was markedly reduced in Serpine1Tg organoids. FACS analysis revealed a significant suppression in the number of AT2 cells, in particular, the CD44+ subpopulation, in Serpine1Tg organoids. A lesser ratio of AT1:AT2 cells in Serpine1Tg organoids was observed compared with wt cultures. There was a significant increase in transepithelial resistance but not amiloride inhibition.ConclusionsOur study suggests elevated PAI-1 in injured lungs downregulates alveolar epithelial regeneration by reducing the AT2 self-renewal, particularly in the CD44+ cells.

Passaging Primary Human Bronchial Epithelia Reduces CFTR-Mediated Fluid Transport and Alters mRNA Expression.

Primary human bronchial epithelial cultures (HBECs) are used to study airway physiology, disease, and drug development. HBECs often replicate human airway physiology/pathophysiology. Indeed, in the search for cystic fibrosis (CF) transmembrane conductance regulator (CFTR) therapies, HBECs were seen as the "gold standard" in preclinical studies. However, HBECs are not without their limitations: they are non-immortalized and the requirement for human donors, especially those with rare genetic mutations, can make HBECs expensive and/or difficult to source. For these reasons, researchers may opt to expand HBECs by passaging. This practice is common, but to date, there has not been a robust analysis of the impact of expanding HBECs on their phenotype. Here, we used functional studies of airway surface liquid (ASL) homeostasis, epithelial barrier properties, and RNA-seq and Western blotting to investigate HBEC changes over two passage cycles. We found that passaging impaired CFTR-mediated ASL secretion and led to a reduction in the plasma membrane expression of the epithelial sodium channel (ENaC) and CFTR. Passaging also resulted in an increase in transepithelial resistance and a decrease in epithelial water permeability. We then looked for changes at the mRNA level and found that passaging significantly affected 323 genes, including genes involved in inflammation, cell growth, and extracellular matrix remodeling. Collectively, these data highlight the potential for HBEC expansion to impact research findings.

The effects of zinc supplementation on primary human retinal pigment epithelium

Population-based and interventional studies have shown that elevated zinc levels can reduce the progression to advanced age-related macular degeneration. The objective of this study was to assess whether elevated extracellular zinc has a direct effect on retinal pigment epithelial cells (RPE), by examining the phenotype and molecular characteristics of increased extracellular zinc on human primary RPE cells. Monolayers of human foetal primary RPE cells were grown on culture inserts and maintained in medium supplemented with increasing total concentrations of zinc (0, 75, 100, 125 and 150 μM) for up to 4 weeks. Changes in cell viability and differentiation as well as expression and secretion of proteins were investigated. RPE cells developed a confluent monolayer with cobblestone morphology and transepithelial resistance (TER) >200 Ω*cm2 within 4 weeks. There was a zinc concentration-dependent increase in TER and pigmentation, with the largest effects being achieved by the addition of 125 μM zinc to the culture medium, corresponding to 3.4 nM available (free) zinc levels. The cells responded to addition of zinc by significantly increasing the expression of Retinoid Isomerohydrolase (RPE65) gene; cell pigmentation; Premelanosome Protein (PMEL17) immunoreactivity; and secretion of proteins including Apolipoprotein E (APOE), Complement Factor H (CFH), and High-Temperature Requirement A Serine Peptidase 1 (HTRA1) without an effect on cell viability. This study shows that elevated extracellular zinc levels have a significant and direct effect on differentiation and function of the RPE cells in culture, which may explain, at least in part, the positive effects seen in clinical settings. The results also highlight that determining and controlling of available, as opposed to total added, zinc will be essential to be able to compare results obtained in different laboratories.

NOD2 Deficiency Impairs the Intestinal Tight Junction Barrier in a Murine Short Bowel Model.

Introduction: Short bowel syndrome with chronic intestinal failure occurs after extensive small bowel resection. Despite intestinal adaptation, protein, energy, fluid and micronutrient balance cannot be maintained by a conventional diet. NOD2 mutations are a risk factor for chronic intestinal failure independent of the underlying disease. The mechanisms are not well understood. In a murine short bowel model the epithelial barrier function in the adapted jejunum was studied in the presence and absence of NOD2. Methods: Male C57BL6/J NOD2 knockout (−/−) and wild type (+/+) mice underwent 40% ileocecal resection (ICR). Sham control mice received transsection of the ileum. Mice were followed up for 14 days. At baseline (d0) 1cm of the most proximal resected ileum was studied in a vertical Ussing chamber and histologically. After 14d 1cm of the most distal remaining small intestine was studied and compared to baseline. In order to study the paracellular leak pathway, flux of 4kDa FITC-dextran was measured. The pore pathway was characterized by transepithelial resistance (TER) and NaCl diultion potentials. Results: Both +/+ and −/− animals showed similar morphologic adaptation 7 days after ICR such as significantly increased villus height (+/+ 229% P=0.028 vs. d0 and −/− 191% P=0.028 vs. d0, n=6-7 each) and crypt depth (+/+ 153% P=0.028 vs. d0 and −/− 129% P=0.028 vs. d0, n=6-7 each). The barrier to uncharged macromolecules (4 kDa FITC-dextran flux) was largely unaffected by NOD2 status. TER was not different in jejunal segments of +/+ and −/− mice at baseline levels (d0) and increased in the adapted jejunum. TER increase at d14 was significantly higher in −/− mice (74.5±6.0 Ω*cm2 +/+ ICR vs. 105.0±10.6 Ω*cm2 −/− ICR, n=5). In line with this, relative permeability to sodium was more severely impaired in the adapted tissue of −/− mice (+/+ ICR d0 7.7±0.4 vs. 14d 6.9±0.3 P=0.35, −/− ICR d0 6.3±0.1 vs. 14d 5.7±0.1 P=0.0157, n=5 each). Conclusion: Profound morphologic adaptation occurs after extensive ileocecal resection. The paracellular leak flux across the adapted epithelium is unaffected by NOD2 status. In contrast, TER is increased after adaptation and more so in absence of NOD2. This may in part be explained by an altered internal electric field in the structurally altered mucosa. However, adaptation of the tight junction barrier occurs as well. Our studies indicate that NOD2 function specifically contributes to the regulation of tight junction mediated charge selective ion flux in the adapted jejunum.

Tumor Necrosis Factor-α Increases Claudin-1, 4, and 7 Expression in Tubular Cells: Role in Permeability Changes.

Tumor necrosis factor-α (TNFα), is a pathogenic cytokine in kidney disease that alters expression of claudins in tubular cells. Previously we showed that in LLC-PK1 cells TNFα caused a biphasic change in transepithelial resistance (TER) consisting of an early drop and recovery, followed by a late increase. However, the underlying mechanisms and the role of specific claudins in the TER effect remained incompletely understood. Here we sought to define how TNFα affects claudins 1, 4, and 7 in tubular cells and to correlate their changes with the TER effect. We show that TNFα elevates total and surface levels of Cldn-1, 4, and 7, and increases their mRNA expression through the ERK and JNK pathways. Further, JNK is also important for TNFα-induced changes in claudin-2 expression. Continuous monitoring of TER using Electric cell-substrate impedance sensing (ECIS) reveals that the two phases of the TNFα effect are differently regulated. Specifically, inhibition of the ERK or JNK pathways prevent the late TER increase, but not the early TER effect. Silencing experiments also show that Cldn-1 is necessary for the early TNFα-induced TER change, while all three claudins appear to contribute to the late TER increase. In summary, we define a central role for ERK and JNK in TNFα-induced altered claudin expression and barrier tightening. Together, our current and previous works show that the TNFα-induced early TER effect requires claudin-1, while claudin-2 decrease is a significant mediator of the late TER increase, and elevation in claudin-1, 4, and 7 contribute to a smaller extent. J. Cell. Physiol. 232: 2210-2220, 2017. © 2016 Wiley Periodicals, Inc.

Effects of Ex Vivo Infection with ETEC on Jejunal Barrier Properties and Cytokine Expression in Probiotic-Supplemented Pigs.

Enterotoxigenic Escherichia coli (ETEC) strains are involved in piglet post-weaning diarrhea. Prophylactic measures including probiotics have been examined in infection experiments with live piglets. In the present study, we have tested whether the early effects of ETEC infection can also be evoked and studied in a model in which ETEC is added to whole mucosal tissues ex vivo, and whether this response can be modulated by prior supplementation of the piglets with probiotics. Jejunal barrier and transport properties of Enterococcus faecium-supplemented or control piglets were assessed in Ussing chambers. Part of the epithelia was challenged with an ETEC strain at the mucosal side. Fluxes of fluorescein as a marker of paracellular permeability, and the expression of selected tight junction (TJ) proteins and of proinflammatory cytokines were measured. The addition of ETEC ex vivo induced an increase in transepithelial resistance peaking in the first 2h with a concomitant reduction in fluorescein fluxes, indicating tightening effects on barrier function. The response of short-circuit current after stimulation with PGE2 or glucose was reduced in epithelia treated with ETEC. ETEC induced a decrease in the TJ protein claudin-4 in the control diet group after 280min and an increase in the mRNA expression of the proinflammatory cytokines interleukin-8 and TNF-α in both groups after 180min. The addition of ETEC ex vivo affected barrier function and transport properties of the jejunal tissues and enhanced cytokine expression. The differences in claudin-4 expression in the jejunum might indicate a beneficial effect of E. faecium prefeeding.

Myrrh exerts barrier-stabilising and -protective effects in HT-29/B6 and Caco-2 intestinal epithelial cells.

Myrrh, the oleo-gum resin of Commiphora molmol, is well known for its anti-inflammatory properties. In different animal models, it protected against DSS-, TNBS- and oxazolone-induced colitis. To date, no information concerning the effect of myrrh on barrier properties are available. Thus, this study investigates the effect of myrrh on paracellular barrier function in the absence or presence of the pro-inflammatory cytokine TNFα. Monolayers of human colon cell lines HT-29/B6 and Caco-2 were incubated with myrrh under control conditions or after challenge with the pro-inflammatory cytokine TNFα. Barrier function was analysed by electrophysiological and permeability measurements, Western blotting, immunostaining in combination with confocal microscopy, and freeze-fracture electron microscopy. In Caco-2 cells, myrrh induced an increase in transepithelial resistance (TER) which was associated with downregulation of the channel-forming tight junction (TJ) protein claudin-2 via inhibition of the PI3 kinase signalling pathway. In HT-29/B6 cells, myrrh had no effect on barrier properties under basic conditions, but protected against barrier damage induced by TNFα, as indicated by a decrease in TER and an increase in fluorescein permeability. The TNFα effect was associated with a redistribution of the sealing TJ protein claudin-1, an increase in the expression of claudin-2 and a change in TJ ultrastructure. Most importantly, all TNFα effects were inhibited by myrrh. The effect of myrrh on claudin-2 expression in this cell line was mediated via inhibition of the STAT6 pathway. This study shows for the first time that myrrh exerts barrier-stabilising and TNFα-antagonising effects in human intestinal epithelial cell models via inhibition of PI3K and STAT6 signalling. This suggests therapeutic application of myrrh in intestinal diseases associated with barrier defects and inflammation.

Tumor necrosis factor-α induces a biphasic change in claudin-2 expression in tubular epithelial cells: role in barrier functions.

The inflammatory cytokine tumor necrosis factor-α (TNF-α) is a pathogenic factor in acute and chronic kidney disease. TNF-α is known to alter expression of epithelial tight junction (TJ) proteins; however, the underlying mechanisms and the impact of this effect on epithelial functions remain poorly defined. Here we describe a novel biphasic effect of TNF-α on TJ protein expression. In LLC-PK1 tubular cells, short-term (1-6 h) TNF-α treatment selectively elevated the expression of the channel-forming TJ protein claudin-2. In contrast, prolonged (>8 h) TNF-α treatment caused a marked downregulation in claudin-2 and an increase in claudin-1, -4, and -7. The early increase and the late decrease in claudin-2 expression involved distinct mechanisms. TNF-α slowed claudin-2 degradation through ERK, causing the early increase. This increase was also mediated by the EGF receptor and RhoA and Rho kinase. In contrast, prolonged TNF-α treatment reduced claudin-2 mRNA levels and promoter activity independent from these signaling pathways. Electric Cell-substrate Impedance Sensing measurements revealed that TNF-α also exerted a biphasic effect on transepithelial resistance (TER) with an initial decrease and a late increase. Thus there was a good temporal correlation between TNF-α-induced claudin-2 protein and TER changes. Indeed, silencing experiments showed that the late TER increase was at least in part caused by reduced claudin-2 expression. Surprisingly, however, claudin-2 silencing did not prevent the early TER drop. Taken together, the TNF-α-induced changes in claudin-2 levels might contribute to TER changes and could also play a role in newly described functions of claudin-2 such as proliferation regulation.

Histatin-1, a histidine-rich peptide in human saliva, promotes cell-substrate and cell-cell adhesion.

Histatins (Hsts) are histidine-rich peptides exclusively present in the saliva of higher primates. In this study, we explored the effects of Hsts on cell-substrate and cell-cell adhesion. Histatin (Hst)-1 caused a significant (>2-fold) increase (EC50 = 1 µM) in the ability of human adherent cells to attach and spread, even in conditions that impaired cell spreading. Other tested Hsts did not stimulate cell spreading, indicating a specific effect of Hst1. The effect of Hst1 on cell-cell adhesion was investigated by using transepithelial resistance (TER) measurements in the human cell line Caco-2, a widely used model for the epithelial layer. We found that 10 µM Hst1 caused a 20% increase in TER compared to the negative control, indicating a function for Hst1 in intercellular cell adhesion and epithelial integrity. A role for Hst1 in both cell-substrate and cell-cell adhesion is highly conceivable, because these 2 modes of adhesion are closely related via shared components and connected signaling pathways.

The relative balance of GM-CSF and TGF-β1 regulates lung epithelial barrier function.

Lung barrier dysfunction is a cardinal feature of the acute respiratory distress syndrome (ARDS). Alcohol abuse, which increases the risk of ARDS two- to fourfold, induces transforming growth factor (TGF)-β1, which increases epithelial permeability and impairs granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent barrier integrity in experimental models. We hypothesized that the relative balance of GM-CSF and TGF-β1 signaling regulates lung epithelial barrier function. GM-CSF and TGF-β1 were tested separately and simultaneously for their effects on lung epithelial cell barrier function in vitro. TGF-β1 alone caused an ∼ 25% decrease in transepithelial resistance (TER), increased paracellular flux, and was associated with projections perpendicular to tight junctions ("spikes") containing claudin-18 that colocalized with F-actin. In contrast, GM-CSF treatment induced an ∼ 20% increase in TER, decreased paracellular flux, and showed decreased colocalization of spike-associated claudin-18 with F-actin. When simultaneously administered to lung epithelial cells, GM-CSF antagonized the effects of TGF-β1 on epithelial barrier function in cultured cells. Given this, GM-CSF and TGF-β1 levels were measured in bronchoalveolar lavage (BAL) fluid from patients with ventilator-associated pneumonia and correlated with markers for pulmonary edema and patient outcome. In patient BAL fluid, protein markers of lung barrier dysfunction, serum α2-macroglobulin, and IgM levels were increased at lower ratios of GM-CSF/TGF-β1. Critically, patients who survived had significantly higher GM-CSF/TGF-β1 ratios than nonsurviving patients. This study provides experimental and clinical evidence that the relative balance between GM-CSF and TGF-β1 signaling is a key regulator of lung epithelial barrier function. The GM-CSF/TGF-β1 ratio in BAL fluid may provide a concentration-independent biomarker that can predict patient outcomes in ARDS.

Aldosterone Upregulates Tight Junction Expression of Claudin‐4, Decreasing Paracellular Na+ Permeability in the Renal Cortical Collecting Duct

Under conditions of low dietary salt intake, plasma aldosterone levels are upregulated, increasing Na+ reabsorption in the renal cortical collecting duct (CCD). While aldosterone is known to enhance transcellular Na+ reabsorption, its effect on paracellular transport remains unknown. Paracellular transport is mediated by tight junction (TJ) proteins called claudins. We used a murine CCD cell line, mpKCCD c14, to study the effect of aldosterone on paracellular transport. We found that aldosterone significantly increased transepithelial resistance (TER) and decreased paracellular Na+ permeability (PNa), when transcellular transport is blocked. We also found that aldosterone increased TJ expression of a Na+ barrier claudin, claudin‐4, suggesting that increased claudin‐4 expression at the TJ is responsible for the decrease in PNa. To confirm this, we stably knocked–down claudin‐4 expression using shRNAs. This diminished both baseline TER and aldosterone‐induced increases in TER, and PCl/PNa.Aldosterone is known to signal via a mineralocorticoid receptor and activate a serum glucocorticoid kinase (Sgk1) to enhance ENaC activity. We found that aldosterone induced changes in claudin‐4 expression and TER were also suppressed by treatment with RU486 (a steroid receptor blocker) and GSK650394 (aSgk1 antagonist). In summary, our data strongly suggests that aldosterone acts via a Sgk1 mediated pathway to upregulate expression of claudin‐4 at the TJ in the renal CCD, decreasing PNa, preventing back‐leak of actively reabsorbed Na+, and increasing net salt reabsorption. Consistent with our in‐vitro data, we have also shown that in mice fed a low salt diet renal claudin‐4 expression is enhanced.

Deoxycholic acid induced changes in electrophysiological parameters and macromolecular permeability in murine small intestine with and without functional enteric nervous system plexuses.

We have previously shown in mice that the fecal proportion and concentration of the hydrophobic bile acid deoxycholic acid (DCA) is elevated with high-fat feeding and that these changes are able to disrupt the intestinal barrier function. The aim of this study was to investigate whether these changes are mediated by the enteric nervous system (ENS). The function of the ENS in the small intestinal tissues of mice was compromised by two different methods: by removing the seromuscular layer and by incubating the intact tissues with tetrodotoxin (TTX), a neural conduction blocker, before DCA treatment. Tissues with or without functional plexuses were mounted into a Ussing chamber system and treated with 3mM DCA for 20min. After DCA treatment, the intestinal permeability to fluorescein was assessed. Short-circuit current (Isc ) and transepithelial resistance (TER) were recorded throughout the experiment. DCA increased intestinal fluorescein permeability only in tissues where the seromuscular layer was removed. In tissues with intact seromuscular layer, DCA induced a significant increase in TER, which was attenuated by blocking of the neural function by TTX. The results of this study suggest that the DCA-induced increase observed in fluorescein permeability is not mediated through neural pathways, but more due to a direct effect on the epithelium. However, as TTX was able to attenuate the DCA-induced increase in TER, it can be speculated that DCA is also able to elicit responses through neural pathways.

The mechanism of serine protease‐induced increases in epithelial barrier function in intestinal epithelial cells (60.7)

Apical serine proteases increase barrier function in intestinal epithelial cells (IECs). We investigated the requirement for proteolytic, protein kinase C (PKC)ζ, and epidermal growth factor receptor (EGFR) activities in the prolonged transepithelial resistance (TER) response to trypsin in IECs.Methods. 1. SCBN epithelial cell monolayers grown on Transwells were treated with trypsin (45BAU/ml) and collected for measurement of phospho‐PKCζ at 2, 5, 10, 15, 30, and 60 min using Western blot. 2. TER across SCBN monolayers was measured in Ussing chambers. Trypsin activity was inhibited by soybean trypsin inhibitor (SBTI) treatment at 10 min (start of plateau phase of TER response) or 30 min (after plateau phase had been established). 3. To investigate the role of EGFR, cells were pretreated with an EGFR inhibitor (PD15305) before addition of trypsin.Results. 1. Upregulation of phospho‐PKCζ was observed in trypsin‐treated SCBNs for up to 60 min. 2. Apical trypsin caused an increase in TER that reached a sustained plateau by 10 min. SBTI at 30 or 60 μg/ml decreased the trypsin‐induced increase in TER by 65% and 80% (respectively) at 10 min and by 70% and 80% at 30 min. 3. Inhibition of EGFR with PD153035 reduced the trypsin‐induced TER by 55%.Our data show the requirement for ongoing proteolytic activity, sustained PKCζ activation and EGFR activity in the trypsin‐induced increase in intestinal epithelial barrier function.Grant Funding Source: Supported by the Canadian Institutes of Health Research

Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins

Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.

TcpC protein from E. coli Nissle improves epithelial barrier function involving PKCζ and ERK1/2 signaling in HT-29/B6 cells.

The probiotic Escherichia coli Nissle 1917 (EcN) is widely used to maintain remission in ulcerative colitis. This is thought to be mediated by various immunomodulatory and barrier-stabilizing effects in the intestine. In this study, the mechanisms of barrier modulation by EcN were studied in the human epithelial HT-29/B6 cell culture model.EcN supernatant increased transepithelial resistance (TER) and reduced permeability to mannitol because of sealing of the paracellular passage pathway as revealed by two-path impedance spectroscopy. This increase in TER was attributed to the TcpC protein of EcN. TcpC induced protein kinase C-ζ (PKCζ) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which in turn resulted in upregulation of the barrier-forming tight junction protein claudin-14. By specific silencing of protein expression by small interfering RNA (siRNA), the sealing function of claudin-14 was confirmed. In conclusion, the TcpC protein of EcN affects innate immunity by improving intestinal barrier function through upregulation of claudin-14 via PKCζ and ERK1/2 signaling.

Liver X receptor agonists decrease ENaC-mediated sodium transport in collecting duct cells

Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol, fatty acid, and glucose metabolism in various tissues. However, the renal action of LXRs is not well understood. Here we investigated the effects of LXR-activating ligands on modulation of epithelial sodium channel (ENaC)-mediated sodium transport in collecting duct cells. Exposure of the M1 cells to the synthetic LXR agonists T0901317 and GW3965 or the natural ligand 22R-hydroxycholesterol for 24 h decreased amiloride-sensitive sodium transport, corresponding with an increase of transepithelial resistance. The inhibition of amiloride-sensitive sodium transport after incubation with T0901317 or GW3965 was not mediated by a reduction of Na(+)/K(+)-ATPase-mediated basolateral sodium transport. On the other hand, T0901317 and GW3965 decreased mRNA abundance and membrane expression of ENaC. Preincubation the monolayer with GW3965 attenuated aldosterone-induced stimulation sodium transport. In primary cultures of collecting duct cells, T0901317 and GW3965 similarly inhibited ENaC transport function as in M1 cells. This is the first evidence showing LXR-activating ligands modulate ENaC-mediated sodium transport in collecting duct cells. These results suggest that LXRs may represent a novel therapeutic target for treatment of conditions with dysregulation of ENaC such as hypertension.

Interleukin-6 Modulates Colonic Transepithelial Ion Transport in the Stress-Sensitive Wistar Kyoto Rat

Immunological challenge stimulates secretion of the pro-inflammatory cytokine interleukin (IL)-6, resulting in variety of biological responses. In the gastrointestinal tract, IL-6 modulates the excitability of submucosal neurons and stimulates secretion into the colonic lumen. When considered in the context of the functional bowel disorder, irritable bowel syndrome (IBS), where plasma levels of IL-6 are elevated, this may reflect an important molecular mechanism contributing to symptom flares, particularly in the diarrhea-predominant phenotype. In these studies, colonic ion transport, an indicator of absorption and secretion, was assessed in the stress-sensitive Wistar Kyoto (WKY) rat model of IBS. Mucosa-submucosal colonic preparations from WKY and control Sprague Dawley (SD) rats were mounted in Ussing chambers and the basal short circuit current (ISC) was electrophysiologically recorded and compared between the strains. Exposure to IL-6 (1 nM) stimulated a secretory current of greater amplitude in WKY as compared to SD samples. Furthermore, the observed IL-6-mediated potentiation of secretory currents evoked by veratridine and capsaicin in SD rats was blunted in WKY rats. Exposure to IL-6 also stimulated an increase in transepithelial resistance in both SD and WKY colonic tissue. These studies demonstrate that the neuroexcitatory effects of IL-6 on submucosal plexi have functional consequences with alterations in both colonic secretory activity and permeability. The IL-6-induced increase in colonic secretory activity appears to neurally mediated. Thus, local increases in IL-6 levels and subsequent activation of enteric neurons may underlie alterations in absorpto-secretory function in the WKY model of IBS.

RhoA and Rho kinase mediate cyclosporine A and sirolimus-induced barrier tightening in renal proximal tubular cells

The regulation and maintenance of the paracellular transport in renal tubular epithelia is vital for kidney functions. Combination of the immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) exerts powerful immunosuppression, but also causes nephrotoxicity. We have previously shown that CsA and SRL elevate transepithelial resistance (TER) in kidney tubular cells partly through MEK/ERK1/2. In this work we examined the hypothesis that the RhoA pathway may also be mediating effects of CsA and SRL. We show that CsA and the CsA/SRL combination activated RhoA, induced cofilin phosphorylation and promoted stress fiber generation. The Rho kinase (ROK) inhibitor, Y27632, prevented CsA and CsA/SRL-induced cofilin phosphorylation and actin remodelling, reduced the TER increase and prevented the rise in claudin-7 levels caused by the drugs. Expression of the exchange factor GEF-H1/lfc was elevated in cells treated with CsA and CsA/SRL. GEF-H1 silencing inhibited RhoA activation by ≈50%, and potently reduced cofilin phosphorylation and stress fiber formation induced by CsA and CsA/SRL. However, GEF-H1 downregulation did not prevent the TER change. Thus the Rho/Rho kinase pathway was involved in mediating CsA and CsA/SRL-induced cytoskeleton rearrangement and TER changes via claudin-7 expression. Our data however point to differential regulation of Rho activation involved in central cytoskeleton remodelling, that is GEF-H1-dependent and junctional permeability that does not require GEF-H1.

Reply from Scott M. O’Grady

The study by Palmer et al. (2011) is the third paper published from our laboratory on the characterization of a human mammary epithelial (HME) cell line that we immortalized by expression of the catalytic subunit of the human telomerase gene (Kim et al. 2002). In our previous study addressing the ion transport properties of these cells (Lee et al. 2007) we showed that the Na+ transport phenotype, P2Y receptor expression profile and lack of anion secretion observed in primary cells matched the results obtained from parallel studies conducted with immortalized HME cells. These findings stand in contrast to isolated mouse and bovine mammary epithelial cells that exhibit both ENaC-dependent Na+ transport and secretagogue-stimulated anion secretion (Blaug et al. 2001, 2003; Schmidt et al. 2001; Quesnell et al. 2007). In response to Dr Shennan's first comment on the origin of HME cells, as stated in our earlier study (Lee et al. 2007), we do not have conclusive proof that the cells originate from the duct. However, HME cells were obtained from a 51-year-old woman, an age where the process of lobular involution significantly reduces the number of secretory alveoli associated with the terminal portions of the ducts (Radisky & Hartmann, 2009). Moreover, lobular alveoli are thought to be the anatomic structures that give rise to breast cancer and in human mammary epithelial cancer cells (MCF-7 cells), purinergic stimulation increases CFTR-dependent anion transport (Flezar & Heisler, 1993). In contrast, HME cells do not exhibit anion secretion in response to purinergic receptor stimulation or to increases in intracellular Ca2+ or cAMP, and CFTR expression is extremely low; thus HME cells do not possess transport characteristics that are typically associated with fluid-secreting epithelia. However, ENaC-dependent Na+ absorption is characteristic of duct epithelial cells found in other exocrine glands such as salivary glands and sweat ducts (Cook et al. 2002; Reddy & Quinton, 2005). With respect to the second comment raised in Dr Shennan's letter, we would like to point out that the experiments described in Palmer et al. (2011) and Lee et al. (2007) were conducted with HME cells grown in media containing hydrocortisone, insulin, epidermal growth factor and bovine pituitary extract as a source for lactogenic hormones. Information about the composition of mammary epithelial growth media (MEGM) is explicitly stated in the Materials section of the manuscript. Our results showing that stimulation with ATP or UTP produces mobilization of intracellular Ca2+ is entirely consistent with what is known about P2Y receptor signalling pathways and is similar to the results obtained with ATP stimulation of MCF-7 cells (Flezar & Heisler, 1993). We suggest that the lack of Ca2+ mobilization following ATP stimulation in lactating mouse acini and bovine mammary cells referenced by Dr Shennan may reflect species differences that could be related to distinct P2Y receptor expression patterns and/or second messenger coupling. As for Dr Shenann's third comment about the previous studies by Linzell and Peaker (Linzell & Peaker, 1971a,b;), it is certainly possible that the transport characteristics of HME cells may not reflect the in vivo ion permeability properties of the duct epithelium. However, it is equally possible that the results obtained by Linzell and Peaker in goats do not necessarily represent the permeability characteristics of human duct epithelium. A possible application of the results obtained from our studies that might provide added insight about the permeability properties of human duct epithelium would be to use ENaC and KCa3.1 channel expression as possible biomarkers for duct epithelial cells. Future experiments could then employ immunohistochemical techniques using sections of human breast tissue to investigate the cellular distribution of these channels, which could be helpful in clarifying the role of the duct in determining the ionic composition of milk. The fourth comment in Dr Shennan's letter suggests an interesting experiment that we have not performed. It would not be surprising that lowering the ionic strength of the apical solution would produce an increase in transepithelial resistance since transcellular ion movements would be reduced under these conditions. However, there may also be effects on expression or activity of membrane transporters, or possibly the paracellular pathway, that could be important in determining the overall permeability of the duct epithelium. Therefore, we would agree that future experiments examining the effects of milk constituents such as lactose on transport function would be worthwhile and perhaps expand the utility of cultured cell models in understanding the regulation of epithelial transport mechanisms that contribute to the ionic composition of milk.

Increase of transepithelial resistance (TER) after removal of Cisplatin from LLC-PK1 monolayers

Increase of transepithelial resistance (TER) after removal of Cisplatin from LLC-PK1 monolayers